Method and device for treating microscopic residual tumors remaining in tissues following surgical resection

ABSTRACT

This invention concerns treating apparently normal tissue surrounding sites of cancerous tumors so as to reduce both the probability of a recurrence of cancer at and near the site of a cancerous tissue, and to reduce the amount of apparently healthy tissue that is usually excised along with the tumor, thereby providing a substantial benefit to the cancer patient by eliminating or delaying tumor recurrence and sparing normal tissue for its functionality and for avoiding unnecessary disfigurement.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/278,721, filed Oct. 21, 2011 which is a divisional of U.S. patentapplication Ser. No. 11/713,181, filed Mar. 2, 2007, which claims thebenefit, under 35 U.S.C. § 119(e), of: U.S. Provisional PatentApplication No. 60/778,740, which was filed on Mar. 3, 2006, was of sametitle and named Paul Goldfarb and Dietmar Rabussay, as inventors. Theentirety of this application and document is incorporated by reference.

FIELD OF THE INVENTION

This invention relates to electroporation systems, devices and methodsof using such devices for treating tissues surrounding sites of tumors.More specifically, this invention relates to the debulking of tumormasses, sparing of tissue surrounding tumors and reducing tumor masssize and recurrence rates by treating apparently non-cancerous tissueswith electroporative pulses and anticancer agents.

BACKGROUND OF THE INVENTION

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that anysuch information is prior art, or relevant, to the presently claimedinventions, or that any publication specifically or implicitlyreferenced is prior art.

Except for vascular disease, cancer is the most frequent cause of deathin industrialized countries. The traditionally accepted paradigm fortreating cancers and tumors that comprise a single relatively welldefined tissue mass has included the use of radiation and chemotherapytypically in association with surgical removal. In cases where surgicalresection is an option, surgery is usually the most effective form oftreatment. However, the effectiveness of surgical treatment depends onthe complete removal of malignant tissue, encompassing the main tumormass as well as branches and micrometastases which are frequentlypresent in the vicinity of the main tumor.

When removing the main tumor, the surgeon also attempts to eliminatesuch local or regional micrometastases by resecting apparently normaltissue surroundings the tumor. That tissue is referred to as “margintissue” or simply as “margin.” To what extent margin tissue is removedis subject to the surgeon's judgment. Typically, a margin of 0.5-2 cmaround the entire tumor is acceptable. However, more extensiveresections are not uncommon for invasive tumors. Even after carefulresection of the tumor and margin, most types of tumors recur with afrequency of 10-40%. The recurrence rate depends on multiple factorsincluding tumor size, type and location of the tumor, condition of thepatient, etc. In order to reduce the rate of recurrence, surgery isusually followed by radiation and/or chemotherapy. Despite suchsecondary treatments recurrence rates are still uncomfortably high.

In addition to surgery and radiation, other methods of local tumorcontrol are in use. These include Radiofrequency (RF) Ablation,Photodynamic Therapy (PDT), Cryotherapy (CRYO), Chemo-Radiation (CR),Brachytherapy (BT), Galvanotherapy (GT), and others. Surgery, RF, PDT,CRYO and CR rely on the complete removal or destruction of the tumor andmargin tissues, whereas radiation, BT, and GT leave treated normaltissue more or less intact, although radiation and BT may cause severescarring, fibrosis and vascular and neural damage. In any event,removal, scarring, and physical damage of healthy tissue can result insubstantial disfigurement and substantial loss of physical use of bodyparts and/or functionality thereof. For example, most of the abovelisted current adjuvants to surgery for destroying or removing tumormasses cause nonspecific damage to normal tissues surrounding the tumor.Excision of a tumor mass can be completely debilitating to functionwhere tumors must be removed from organs including the tongue, vocalchords, rectum, labia, penis, or to fine muscle and visual structures ofthe face tissue.

To avoid such disfigurement and preservation of function and to ensurethat tissues surrounding the tumor are cleared of such cancerous cells,the present invention is provided as an adjuvant or neo-adjuvant tosurgery as there is still a need in the oncology arts for a device andmethod of sparing apparently healthy tissues that lie adjacent to tumorsand to reduce tumor mass, growth of the tumor, and recurrence rates.

SUMMARY OF THE INVENTION

In a first embodiment, the invention comprises a method of reducing theprobability of recurrence of cancer cell growth in a tissue. In apreferred embodiment, the method includes providing to the cells of saidtissue both an electroporating pulse of electric energy and amedicament. In a related embodiment the medicament is preferablyprovided to the tissue immediately prior to or simultaneously with theelectroporating pulse.

In a second embodiment, the invention comprises a method of treatingresidual cancerous cells remaining in tissues following surgicalresection. Preferably, the invention provides for controlling furtherspreading of cancer by subjecting microscopic nodules or other forms ofcancerous tissue to the medicament in an electroporating electric field.In a related embodiment, such treatment can be an adjuvant to surgery inthat it can be applied either prior to or after tumor removal. In somecircumstances, especially where the cancerous cells have not yet formedinto a fibrous mass, no surgical procedure may be employed. In suchcase, the EP treatment provides a method to reduce tumor mass andterminate or delay further growth of cancerous cells in the tissue. Instill a further related embodiment, the invention methods provide fordebulking of larger tumor masses by causing through the effect of ananticancer agent, such as Bleomycin, a ‘softening’ of the tumor tissuesuch that the tumor mass will be easier to remove from surroundinghealthy or normal tissue.

In a third embodiment, the invention method provides for decreasing theamount of normal tissue surrounding a tumor site, i.e. the “margin”tissue, that must be removed at the time of tumor excision and therebyspare normal tissue and consequently provide for greater retention oftissue function, and appearance.

In still other embodiments, the invention provides an instrument that iscapable of providing said electroporating energy pulses to the tissuesurrounding an excised tumor on demand and in an easily operable manner.In related embodiments, the invention device provides for bothadministration of an anti-cancer agent and administration of electricfields sufficient to cause electroporation of the tissues in the marginregion. Further embodiments of the invention device are provided below.

Other features and advantages of the invention will be apparent from thefollowing drawings, detailed description, and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing wound strength as measured by the breakingpoint of healing skin samples treated in vivo with saline alone, salineplus electroporation, Bleomycin alone, and Bleomycin pluselectroporation. Underlying fat and muscle tissue were also treated inthese experiments.

FIGS. 2A-F are micrographs of trichrome stained tissues showingcomparisons between (A) untreated tissue at day 2 post incision, and (B)tissue treated with saline plus electroporation at day 2 post incision,(C) saline plus electroporation at week 2, (D) Bleomycin pluselectroporation at 2 weeks, (E) saline plus electroporation at 3 weeks,and (F) Bleomycin plus electroporation at 3 weeks.

FIGS. 3A-D are stained (Trichrome) micrographs of collagen deposition inskin incisions after three weeks. FIGS. 3A and C are bright field imagesfor Bleomycin and Saline treated skin tissues, respectively, while FIGS.3B and C are polarized light images showing Bleomycin and Saline treatedskin tissues, respectively.

FIGS. 4A-D are micrographs showing samples of muscle tissue at 3 weekspost incision; (A) saline alone, (B) saline plus electroporation, (C)Bleomycin alone, and (D) Bleomycin plus electroporation.

FIG. 5 is a cross sectional figure of one embodiment of the inventiondevice showing an embodiment comprising a handle comprising a thumbwheel for raising and lowering an array of plungers which actuateloading or dispensing of a substance through the electrode needles.

FIG. 6 is a drawing depicting an exploded view of one embodiment of theinvention.

FIG. 7 is a cross sectional drawing of one embodiment of the inventiondevice wherein the plungers are actuated by a screw driven by clockwise(raising plungers) and counter clockwise (lowering plungers) rotation ofthe housing 50.

FIGS. 8A and B are perspective drawings of one embodiment of theinvention wherein rotation of the housing 50 will drive the array ofplungers back and forth. Figure A shows the embodiment with the tray 20while Figure B shows the main body of the embodiment without the tray20.

FIG. 9 is a perspective drawing showing one embodiment of the inventionwherein the array of plungers is actuated via a wing nut.

FIG. 10 shows an example of the array of the plurality of electrodeneedles from the underside of the substrate 22.

FIGS. 11 A, B, C, and Dare photographs of Group 2 cohort animals showingthe test animal with tumor (A), the tumor surgically exposed prior tocomplete tumor removal (B), the open wound bed after tumor removal butprior to sham EP (C), and the surgical/treatment site after 3 weeks posttreatment (D). With this Group 2, no electroporation was performed andthe tumor recurred even though the tumor had been completely removed.

FIGS. 12 A, B, C, and D are photographs of Group 1 cohort animalsshowing the test animal with tumor (A), the tumor surgically exposedprior to complete tumor removal (B), the open wound bed after tumorremoval but prior to treatment with EP (C), and the surgical/treatmentsite after 3 weeks post treatment (D). As observed with Bleomycin-EPtreatment no tumor recurred at the site of treatment.

FIGS. 13 A, B, and C are photographs of Group 7 cohort animals showingthe test animal with tumor (A), after partial tumor removal (B), and thesurgical/treatment site after 3 weeks post treatment (C). The mouse at 3weeks showed no tumor recurrence even though the tumor was onlypartially removed.

FIGS. 14 A, B, and C are photographs of Group 4 cohort animals showingthe test animal with tumor (A), after partial tumor removal (B), and thesurgical/treatment site after 3 weeks post treatment (C). Without EPTthe tumor continued to grow.

FIG. 15 shows a bar graph of the data presented in Table V. PTE refersto partial tumor excision, CTE refers to complete tumor excision, Bi.v.means Bleomycin administered to test animals intraveneously, EP meanselectroporation, i.t.B refers to Bleomycin administered intratumorally,and PEP means partial electroporation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As those in the art will appreciate, the following description describescertain preferred embodiments of the invention in detail, and is thusonly representative and does not depict the actual scope of theinvention. Before describing the present invention in detail, it isunderstood that the invention is not limited to the particular devicearrangements, systems, and methodologies described, as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the invention defined by the appended claims.

Overview

Electroporation Therapy (EPT), also known as Electrochemotherapy (ECT),is a method to treat localized cancerous lesions and tumor masses. Themethod comprises administering certain chemotherapeutic drugs, mostcommonly Bleomycin, either intratumorally or intravenously. In mostcases electroporation (EP) is performed by inserting arrays of needleelectrodes and delivering pulsed electrical fields emanating from theseelectrodes directly to the cancerous cell mass. Pulse parameters aregenerally within the following ranges: field strength 200-2000 V/cm;pulse length 0.1-10.0 ms; pulse number 2-20; and pulse frequency 1-5 Hz.Applying such electric fields results in permeabilization of tumor cellmembranes which allows anticancer drugs to enter cells and to cause upto 5000-fold greater cytotoxicity due to the drug uptake than observedin the absence of electroporation. EPT has been shown to be effectiveagainst many types of solid tumors in animals and several types oftumors in humans. In fact, several clinical studies are presentlyongoing, including a Phase III study evaluating the safety and efficacyof EPT for the treatment of squamous cell carcinomas of the head andneck. However, such treatment studies are not designed or intended totreat or test the affect of EPT on noncancerous margin tissue coincidentto cancerous cells.

EPT is somewhat unique among the many methods employed for local andregional tumor control in that it is far less damaging to normal tissuethan to malignant tissue. Results from in vitro as well as in vivoanimal and human studies indicate that EPT using a drug, such asBleomycin, effectively destroys most tumors via apoptotic and necroticmechanisms while causing only a minor effect (inflammation, minornecrosis) on normal tissues surrounding the tumor, particularly whenBleomycin concentration, doses, and pulse parameters are withinappropriate ranges. The margin tissue surrounding tumors that istypically amenable to treatment include, without limitation, those oforgans including breast, prostate, tongue, penis, labia, rectum, vocalchords, liver, squamous cell carcinoma of the head and neck (SCCHN),cutaneous and other tumors. EPT can be performed on such tissues whetherthe tumor mass has been removed or not. In such cancers the patientwould be helped substantially by treating the tissues in a mannerwherein healing can proceed largely unimpaired and there is apossibility of eliminating microscopic metastases and tumor branchestrending into the tissues that give rise to local recurrences. As isalso understandable with respect to the invention methods,electroporation treatments with agents such as Bleomycin provide for ameasurable degree of selectivity in targeting cancerous cells. Thismethodology is also pertinent to treatments with other agents that havespecific activities against particular disease states, such as highlylocalized infectious diseases and other disease states where there arefoci of infected or diseased tissues wherein a cell, or group of cellsthat are infected or diseased, can be targeted by the agent to destroythe affected cells. In such case electroporation of such cells andsurrounding tissue will allow the agent to reach its intended target inthe infected or diseased cells, yet not have a detrimental effect onuninfected or normal cells as empirically observed. The molecular andcellular mechanisms underlying the different response of abnormal andnormal cells, respectively, is presently unknown. Additionally, use ofthe invention methods and device is applicable to disease states wheresites of cancerous cells are distributed in areas not easily amenable tosurgery. For example, skin tissues of the face containing microscopic orfocal disease sites can be treated with the invention device in the samemanner as used for treating a margin tissue bed resulting insubstantially less scarring than would be caused by surgery. Thus, theinvention methods can be used as an adjuvant to surgery or even aneo-adjuvant. Specifically, cells of tumor mass can be electroporatedalong with surrounding normal tissue and thereby provide for a method ofdebulking the tumor mass in providing for a mechanism to hinder,terminate or otherwise reduce the growth and/or recurrence rates of thediseased cells. In such case, tumor tissue can be removed either beforeor after EPT, or can even be allowed to remain unexcised following EPT,in which cases the effect of an agent such as Bleomycin will provide forcell death by apoptotic and/or necrotic mechanisms and softening of thetumor cell mass followed by further necrosis. Normal tissue in thevicinity will remain largely unaffected.

Typically, treatment of a tumor with EPT includes a Bleomycin dose of 1Unit/ccm tissue to be treated, injected intratumorally at aconcentration of 4 Units/ml. The pulsed fields are generated such thatthe field is usually applied in six separate pulses at 4 Hz, one each in6 different field orientations, each of 100 usec duration, at a nominalfield strength of anywhere between 200 and 2000V/cm but, generallybetween 600 and 1500 V/cm, more usually between 600 and 1400 V/cm andeven more usually between 1200-1300 V/cm across the tumor mass.Increasing Bleomycin dose or concentration and increasing the intensityof the electroporating pulse beyond the standard parameters can resultin more severe effects on normal tissue. At very high field strengthsand/or pulse lengths EP by itself (i.e., for example, without Bleomycin)can induce irreversible cell membrane deterioration, leading to thedestruction of tumor as well as normal cellular tissue falling withinthe effective electric field. In still further related embodiments, adevice for operating the methods of the invention can itself be operatedusing pulsed field strengths of between 1-600 V/cm when injecting DNA asa therapeutic agent as set forth in U.S. Pat. No. 6,528,315 whichreference is herein incorporated in its entirety by reference. In suchembodiment, the DNA ideally codes for a polypeptide that provides for atherapeutic anticancer effect on the tumor-containing tissue beingtreated.

With regard to other agents, including genes, proteins, cytokines,chemokines, steroids, antibodies, RNAi, and antisense nucleic acids, thesame high pulsing parameters can be used (i.e., for example, 800-1500V/cm or, alternatively, 1-600 V/cm, more preferably 200 to 600 V/cm, andstill more preferred 400-600 V/cm. It is further contemplated that theseand any other agents can be used where such agents promote wound healingwithout promoting growth of malignant cells. With regard to using suchagents in EPT, lower-end field strengths as low as 50V/cm can be used toelectroporate cells within tissue containing microscopic lesions anddiseased cells where the tissues have not formed an abnormal tissuemass.

As mentioned earlier, the present rationale for cancer therapy involvesin one embodiment surgical removal of the tumor mass (‘debulking’) toreduce biological stress on the patient, surgical elimination of margintissue to eradicate micrometastases and invading tumor tissue, andradiation and/or chemotherapy to control local, regional and systemicspread of the tumor. While surgical removal of the tumor itselfgenerally provides relief for the patient, without severe sequelae, thesubsequent steps of margin resection, radiation and chemotherapy can bevery detrimental to the patient. Physiological and mechanical function,look, quality of life and eventual outcome can be severely affected bythese treatments. Therefore, it would be ideal to combine surgicalremoval of the tumor with a treatment method that (a) prevents tumorrecurrence due to micrometastases and invasive tumor tissue moreeffectively than present methods, and (b) minimizes the loss ofsurrounding margin tissue. In the present invention, a novel applicationof EPT as a surgical adjuvant or neo-adjuvant is provided to achieveeither reduction of tumor mass, prevention of tumor recurrence andminimizing the need to excise margin tissue or both.

Turning now to the invention method, the current invention is intendedto provide a novel method for lowering the probability of recurrence oftumor growth in otherwise normal tissues surrounding a site of excisedcancerous cells. In other words, the invention method comprises treating“margins” of tissue surrounding a site of cancer cells, such cancerouscells typically formed at a distinct tissue site. In a preferredembodiment, the invention method provides for reducing the amount oftissue that must be excised along with the tumor and its cancerous cellsand making radiation or chemotherapy superfluous. The method comprisesapplying an electroporative pulse of electric energy to the tissuessurrounding the tumor site. In addition to the application of anelectroporative pulse, the method further comprises providing,preferentially either prior to or simultaneously with the electricpulse, a formulation comprising an anticancer medicament. In a preferredembodiment, such medicament comprises a biologically active molecule.For example, the medicament can comprise a nucleic acid encoding anexpressible polypeptide, such as, for example, a nucleic acid in anexpression vector and comprising a gene sequence for a cytokine orchemokine, antibody, or enzyme. Another example comprises antisense DNAor RNA, or interfering RNA (RNAi). Alternatively, the medicament cancomprise a polypeptide or an organic molecule such as, for example, acytokine, chemokine, antibody, Cisplatin or Bleomycin, or any othermolecule having an anticancer activity. Further, any of such compoundsdisclosed above can be administered in a formulation that can compriseany combination of pharmaceutically acceptable salts, buffers and otherexcipients as are well known in the art. For example, formulations fornucleic acids can comprise, for example, said nucleic acid andpoly-glutamic acid (poly-L-glutamate) as described in U.S. Pat. No.7,173,116 herein incorporated in its entirety by reference.

In a further embodiment, the invention method comprises applying theelectroporative pulse using a device capable of providing suchelectroporative pulses to localized areas at the tumor site. In apreferred embodiment such a device can include an array of elongateneedle-like electrodes. Alternate embodiments can use shortnon-penetrating electrodes or semi-penetrating microneedle electrodesdepending upon the strength of the electric pulse required and upon thedelivery mode of anticancer agent. In such embodiment, the device wouldbe designed without a need for a plunger and delivery needles. Instead,the treatment methods can be applied using an electroporation devicecomprising an electrode array only, such array comprising thenon-penetrating or semi-penetrating electrodes. In such a case, drugwould be delivered by separate means such as a syringe. In a furtherpreferred embodiment with respect to any of the physical arrangements ofthe invention device, during performance of the treatment procedure, theelectrodes are positionable with respect to the tissues to be treated tocreate an electric field having a field strength and energy sufficientto electroporate cells in said tissue within a specified area and depth.Typically, the device will be positioned to impart an electroporativepulse to all tissues within a preselected margin of tissue around (inthree dimensions) the tumor excision site. Depending upon the size ofthe excised tumor and the size of the electrode array, the surroundingmargin tissue bed can be electroporated either entirely in oneelectroporative pulse application or may require a multiplicity ofelectroporative pulse applications by the positioning and repositioningof the electrode array so as to completely encompass the tumor sitemargin tissue in electroporating energy fields. In some embodiments, theelectrodes can serve as delivery needles for the agent intended to beelectroporated into the cells, while in other embodiments, the agent canbe administered to the tissue independently from theadministration/positioning of the electrodes in the tissue.

Additionally, in order to be sure that the invention method is capableof exposing healthy non-cancerous tissues to electroporative pulseswithout substantially damaging said healthy tissues, a study wasconducted on the recovery characteristics of healthy tissue surroundingan open wound after exposure to electroporative pulses. As disclosedbelow, treatment of healthy tissues with electroporative pulses, orelectroporative pulses and an anticancer agent, imparted no measurablesignificant effect to the cells of the treated area and the open woundshealed similar to untreated and/or normal tissues. Since healthy tissuescan be exposed to an electroporative pulse without substantialdetriment, use of electroporation in the treatment of tissuessurrounding a tumor for the purpose of allowing surgeons to excise anarrower margin of healthy tissue is now possible.

General Treatment Procedure

One embodiment of the invention methods is to treat surgical woundmargins or, more generally, tumor margins and in the process (a) achievea superior recurrence rate (i.e., less frequent recurrence) comparedwith conventional surgical tumor and margin resection with or withoutsecondary treatment; (b) reduce or eliminate the need for surgicalresection of margin tissue while maintaining equal or better recurrencerates as with traditional surgical margin removal, and thus preservefunctional tissue; and (c) reduce or eliminate the need for radiation orchemotherapy subsequent to surgical tumor resection (with or withoutmargin resection) while maintaining equal or superior recurrence ratesas with traditional surgical margin resection and secondary treatmentssuch as, e.g., radiation, and chemotherapy.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

Example I

Treatment of Apparently Healthy Tissue Surrounding an Open Wound withElectroporation Pulses and/or Anticancer Agent

In order to successfully use electroporation in areas of open woundscomprising for the most part normal tissue, we conducted a series ofexperiments to show that electroporation of normal tissue, with orwithout anti-cancer agent, can be performed without significantlyaffecting the healing processes in cutaneous, subcutaneous and muscletissues.

In this experiment eighteen transcutaneous incisions were made into thedorsal muscles in each of eight pig test animals grouped 1-6. In studygroups 1-5, longitudinal incisions were made while in group 6 theincisions were transverse to the longitudinal orientation of the musclefiber. At each incision site, either nothing, normal saline, orBleomycin in solution was injected such that injections were placed to a1 cm depth and spaced at 1 cm distances from one another with threeinjections, one on each side of each incision within 3 cm of each otherand one in the center of the incision. Bleomycin, an anti-cancer agent,was used wherein each injection consisted of 0.125 ml (4 units/mlsaline). The incisions were then treated with bipolar pulses of 530 V,100 μsec each, from a pulse generator using a 6-needle electrode arrayof 0.5 cm diameter (nominal field strength for each pulse was 1,233V/cm).

Specifically, 8 male Yorkshire/Hampshire cross swine (40-50 kg) wereacclimated 5-7 days prior to surgery. Incision locations were marked onthe dorsum using sterile surgical markers. Each incision was 5 cm longand made to a depth of 5 mm into the muscle. All animals in groups 1-5received 18 longitudinal incisions each, while animals in group 6received transverse incisions, in accordance with Table I. Each studygroup comprised 24 incisions.

TABLE I Group Incision Injection Electroporation Group 1 (NL)Longitudinal None Group 2 (SL) Longitudinal Saline Group 3 (SEL)Longitudinal Saline X Group 4 (BL) Longitudinal Bleomycin Group 5 (BEL)Longitudinal Bleomycin X Group 6 (BET) Transverse Bleomycin X

Injections of a total volume of 1.5 ml of saline or Bleomycin (6 U) wereadministered at 12 sites per incision, 6 in the muscle and 6 in theskin. Incisions treated with electroporation were electroporated using a0.5 cm diameter, 1.5 cm long needle array. On each side of the muscleincision, electroporation was given 3 times in the central 3 cm of theincision, each electroporation site being 1 cm apart from the other.Skin was not electroporated. The epimysium was closed with 4-0 Vicrylabsorbable interrupted sutures. The skin was closed with 4-0 nyloninterrupted sutures. Swine were sacrificed at 2d, 1 wk, 2 wks, or 3 wksfollowing surgery. Histology specimens containing skin and at least 5 mmdepth of underlying muscle were taken from the center of the incisionand immediately placed in 10% neutral buffered formalin. An additionalcontrol specimen was taken from non-incised skin on the dorsum. Afterstaining and sectioning, skin, subcutaneous tissue, and muscle weregraded histologically on a 0-3 scale using the following parameters:Healing, Granulations/Fibrosis, PMN's, Lymphocytes, Histiocytes,Necrosis, Hemorrhage and Atypical Cells.

Mechanical testing was performed on strips of skin removed from thecenter of the incision at three different time points, namely, Days 7,14, and 21. Skin strips were wrapped in gauze soaked in saline andtested the same day for incision breaking strength using an Instron 900(Instron Corp. Norwood, Mass.) with a 100 pound load cell run at 0.5in/min. Breaking strength was determined by plotting the stretch versusthe strain for each incision up to the breaking point. Breaking strengthof skin incisions increased over time in Group 1 (no saline orBleomycin) from ˜1800 kPa at 1 week to ˜2500 kPa at 2 weeks and ˜5000kPa at 3 weeks post-incision. Injecting saline (Group 2) yielded similarresults. Group 3 (saline injection with electroporation) was notsignificantly different from Group 2 at any time indicating thatelectroporation did not cause adverse effects in breaking strength. At 1week, all three Bleomycin treated groups had significantly (i.e., 90%)reduced breaking strengths compared to all three non-Bleomycin groups,indicating that Bleomycin had delayed dermal wound healing.Electroporation had no adverse effects on breaking strength as Groups 4and 5 were not significantly different from each other. By week 2, therewere no significant differences between Groups 1-5. Only the transverseincisions with Bleomycin and electroporation exhibited lower breakingstrengths. This effect carried through to week 3 where Group 6 hadsignificantly lower breaking strength than Groups 1-5.

As shown in FIG. 1, there was no substantial difference between salinealone, saline plus electroporation, Bleomycin alone, or Bleomycin pluselectroporation after three weeks post incision. Even the strength ofhealed transverse incisions is sufficient not to be of clinical concernin terms of risk to the patient.

Histological Evaluation

Histology specimens at four different time points, namely, Days 2, 7,14, and 21 containing skin and at least 5 mm of muscle were taken fromthe center of the incisions and immediately placed in 10% neutralbuffered formalin. Histological studies showed statistically significantchanges (Bleomycin groups only) present at day 7. All non-treated tissueand saline injected tissue remained normal in appearance. By day 21, allBleomycin injected samples were comparable with saline-injectedcontrols. For example, when comparing saline-injected v.s.saline-injected plus electroporated tissue samples, the only effects ofelectroporation were slight increases in the number of skin histiocytesat 1 week and a reduction in skin lymphocytes at 2 weeks. There were noeffects of electroporation on any of the other parameters such ashealing of skin, subcutaneous tissue and muscle. Further, when comparingBleomycin v.s. Bleomycin plus EP treated tissue samples there were nosignificant differences at 2 days or 1 week. At 2 weeks, electroporationcaused a significant increase in muscle necrosis but that differencedisappeared by 3 weeks. At 3 weeks electroporation caused a reduction inskin histiocytes but showed increased granulation tissue/fibrosis in themuscle, evidence of healing. There were no sustained significantdifferences in healing of skin, subcutaneous tissue or muscle caused byelectroporation even in the presence of Bleomycin.

As shown in FIGS. 2 A-F, wound healing as compared between untreatedtissue, saline plus electroporation treated tissue, and Bleomycin pluselectroporation treated tissue, becomes histologically equivalent byweek three. Specifically, by week three saline plus electroporation isequivalent to Bleomycin plus electroporation (compare E and F). By week2 saline plus electroporation shows more advanced healing than Bleomycinand electroporation (compare C and A). By week 3 saline pluselectroporation appears equivalent to Bleomycin plus electroporation atweek 2 (compare E and D).

With regard to other histological features as shown in FIGS. 3 A-D,collagen is deposited in the healing wound but maybe at a slower ratefor Bleomycin treated samples than samples treated with saline.Specifically, with no electroporation, incisions in saline treatedanimals (C and D) show deep blue collagen fibers in continuity with thecollagen fibers of the dermis while incisions of Bleomycin treatedanimals (A and B) show pale blue collagen fibers with fewer zones ofcontinuity with the dermal collagen. With respect to effect, if any, onthe muscle tissue, as shown in FIGS. 4A-D, at 3 weeks the micrographsindicate that electroporation does not cause significant differences inthe healing of muscle tissue compared to saline or Bleomycin alone.

Considering the effect, if any, of longitudinal vs transverse incisions,in the presence of saline vs Bleomycin, there were no significantdifferences at 2 days or 1 week. At 2 and 3 weeks the only significanthistological differences were in the amount of granulationtissue/fibrosis at all three tissue layers studied. In skin,subcutaneous tissue and muscle, longitudinal incisions had moregranulation tissue/fibrosis than transverse incisions in every casewhere there were significant differences. This is expected aslongitudinal incisions generally heal better than transverse incisions.The scores for healing of skin, subcutaneous tissue and muscle were notsignificantly different for longitudinal and transverse incisions.

SUMMARY

Electroporation of the muscle caused no significant alteration in thebreaking strength of porcine dermis in the presence or absence ofBleomycin at weeks 2 and 3 and arguably even within week 1. However, thepresence of Bleomycin regardless of whether EP was used, was associatedwith a reduction in the breaking strength of porcine dermis up to 1-2weeks post treatment when compared to incision only, or to incisioninjected with saline, with or without EP. With respect to histology,electroporation of the tissues resulted in no significant differences inhealing of the skin, subcutaneous tissue or muscle. Thus,electroporation therapy does not significantly adversely affect healingof muscle or skin. This appears to apply also where EPT is used fortreatment of tissue surrounding tumors, and thus the methods of theinvention provide an alternative to surgical resection or othertherapeutic interventions in the treatment of wound margins aimed atreducing tumor recurrence.

Example II

Hypothetical Treatment Regimen

It is contemplated that surgery using the invention method and devicewill generally follow a protocol likely to employ the following steps:

1. The main tumor mass will be surgically resected as usually practicedby a surgeon skilled in the art. The surgeon will either resect theprimary tumor mass only or will also resect a surgical margin.Alternatively, the main tumor may be ablated by one of many ablativetherapies, such as RF (radio frequency) ablation, PDT (photodynamictherapy), cryotherapy, chemo-radiation, brachytherapy, or evengalvanotherapy, with or without ablation of margin tissue.

2. After surgical resection (or ablation) of the tumor mass, with orwithout resection (or ablation) of margin tissue, the entire tissuesurrounding the resection (or ablation) site will be treated by EPTemploying Bleomycin or other chemotherapeutic or biological drugs.Drug-EPT treatment will be performed to a depth as determined by thesurgeon according to theoretical considerations, scientific studies, orpractical experience.

Variation A. Protocol for Resecting a Tumor with Standard Margins

-   -   1. In this protocol variation, after the tumor has been removed        along with standard margins, the entire wound surface (tumor        margin bed) is treated to an appropriate depth, typically 1 cm        with a standard EPT regimen typically comprising local injection        of an anticancer agent followed by electroporation using at        least a multi electrode array equipped device (for example, a        six needle array or other electrode arrangement of an invention        device such as disclosed in FIGS. 5-10) and, for example,        pulsing in at least three field orientations with an appropriate        voltage. By field orientations is meant the spatial and        directional orientation of the electric field generated by the        electric pulse between any two oppositely charged electrodes. In        this embodiment, the protocol Variation A can be performed        without the use of (or alternatively a reduced use of)        traditional post-surgical secondary treatment. In such instance,        the invention method is intended to achieve equal or better        recurrence rates compared to complete surgical margin resection        and secondary treatment.    -   2. Following EPT of the tumor margin bed, if desired, the        patient can be further treated with conventional secondary        treatment, such as radiation or chemotherapy, and thereby        provide additional potential protection against tumor recurrence        compared with no EPT treatment of the margin tissues.

The outcome of performing steps 1 and 2 above is intended to compriseimproved cure rate or time to recurrence with some degree ofpreservation of tissue over conventional therapies but margin tissuewould have been removed nonetheless.

Variation B. Protocol for Resecting a Tumor with No Margin Resection

-   -   1. In this protocol Variation method, following tumor removal        and reduced or no margin removal, the entire wound surface is        treated to an appropriate depth, typically 1 cm with a standard        EPT regimen typically comprising local injection of an        anticancer agent followed by electroporation using a multi        electrode array (for example, a six needle array or an array as        disclosed in the attached figures) and, for example, pulsing in        at least three field orientations with an appropriate voltage.    -   2. The intended outcome following this protocol and additional        conventional secondary treatment is complete or partial        preservation of margin tissue and potentially improved cure rate        or time to recurrence.

In alternate embodiments of the above protocols, no traditionalsecondary treatment is provided, yet the intended outcome is complete orpartial preservation of margin tissue and improved cure rate or time torecurrence.

Example III

Turning now to a disclosure of an electroporation device suitable totreat margin beds of resected tumors, in a first embodiment the deviceis intended to provide the capability to deliver to the margin tissue ananticancer agent, e.g., for example, Bleomycin, evenly distributedthroughout the margin bed. In a second embodiment, the device providesfor the capability of delivering to the margin tissue a plurality ofpulses of electric energy sufficient to cause electroporation of cellsthroughout the margin bed to a depth of between 1 and 1.5 cm.

In a third embodiment, the invention device can provide for delivery ofthe electroporation pulses to a substantial portion of the margin bed,if not all of the margin bed (depending upon the relative dimensions ofthe device and the tumor), by a single placement of the invention devicesuch that in order to provide electroporating pulses to the entiremargin bed the device should preferably only need to be placed once.

In further embodiments, the device includes a plurality of electrodespositioned in a geometric array to provide for delivery of a series ofelectric pulses between selected electrodes of the array. In a relatedembodiment the device can comprise a plurality of shapes to accommodatevarious tumor shapes and sizes and further provide an array ofelectrodes in a preferred geometry.

In still other embodiments, the electrodes of the invention device cancomprise elongate hollow needles such that the electrodes can act asboth electrodes and anticancer agent delivery needles. In a particularlypreferred embodiment, the invention device can include a sharps cover tocover the electrodes and to provide a mechanism for delivery ofanticancer agent to the hollow needles and a means for keeping theelectrodes sterile prior to surgery.

Turning now to specific details of the invention device, FIG. 5 showsinvention device 10 comprising a substantially rigid sharps cover 20which doubles as a therapeutic agent filling tray. The cover/tray 20further comprises a fill port 21 which can be designed with a fittingcapable of connecting with any desired type fitting for attaching to asource of fluid for filling the tray to a desired level of therapeuticsubstance. The cover/tray 20 snuggly fits the main body of the inventiondevice which itself comprises a substantially rigid substrate 22 throughwhich an array of a plurality of elongate electrodes 23 pass and whichare each individually connected to electric leads 24 which terminate ona lateral portion of the device in a plug or connector 25 for attachmentto a source of electrical energy. The substrate 22 and the plug 25 areconnected to main body substrate 30 which itself comprises an array of aplurality of wells 31 (see FIG. 6). Each well comprises a hollowelectrode/needle through which a substance such as a therapeuticsubstance can be transmitted.

Fitting with the substrate 30 is substrate 40 which comprises an arrayof a plurality of plungers 41. The substrate 40 is connected to amechanism which can actuate the movement of the substrate 40 towards andaway from substrate 30 so as to drive into or extract away fromsubstrate 30 the array of plungers 41 of substrate 40. In oneembodiment, the substrate 40 is actuated by a central rod 60 connectedto substrate 40 on one end and at the other configured into a ratchetand a thumb activated wheel 61. In a related embodiment, substrate 50further forms an enclosing support structure 63 to surround and enclosecentral rod 60. The support structure 63 can further comprise userfriendly handgrip 64. In operation, the operator would use port 21 tofill the cover/tray 20 to a desired level with a fluid therapeuticagent, such as for example Bleomycin, then use the thumb wheel 61 todraw into the wells 31 the agent (FIG. 6). The operator would thenremove the device from the tray 20 using the handle formed by thesupport structure 63 and place the device at an appropriate position onthe tissue or margin bed. The operator would then insert the electrodes,activate the thumb wheel 61 to administer the agent into the tissuefollowed by activation of the electrodes, the device having previouslybeen attached to a source of electrical energy via the plug 25.

The invention device further can comprises other various arrangements ofcomponents to provide the same result. For example, in one embodimentthe device can comprise attached to the substrate 30, an optionallydynamic housing 50. Housing 50 is considered dynamic on an optionalbasis since depending upon the style of the invention chosen, thehousing can rotate clockwise and counter clockwise to raise and lowerthe plunger instead of using a thumb wheel. In embodiments such as thatdepicted in FIG. 5, the housing 50 is not dynamic in that although thehousing 50 may be capable of rotation, but for the handle and thumbwheel, such rotation in this embodiment servers no utility.Alternatively, as depicted in FIGS. 7 and 8, the housing is dynamic inthat substrate 40 is actuated toward or away from substrate 30 by screw70 which is attached at one end to substrate 40 and to housing 50 on theother end by engagement of said screw 70. The housing 50 portion whichinteracts with threads of screw 70 is itself formed into screw threads71 which upon rotation of housing 50 will draw substrate 40 away from ortowards substrate 30. In operation, the operator would fill thecover/tray 20 with agent as previously described, then place the discoiddevice appropriately on the tissue to be treated. After inserting theelectrodes into the tissue, the user would, while pressing down on thedevice, rotate the housing 50 in the direction that will move theplungers toward substrate 30 and thereby expel the agent into thetissues. The operator would then active the electrodes, assuming thedevice had been previously connected by plug 25 to a source ofelectrical energy.

Still other embodiments of the invention are possible, such as forexample use of a wing nut 80 attached to screw 70 for actuating theplungers 41 (see FIG. 9). In such embodiment, the housing 50 would notcomprise screw threads 71 but instead only a smooth bore past which thethreaded central rod 60 can pass. In such embodiment, the operator wouldnot need to rotate the housing but only the wing nut.

Still other embodiments of the invention are possible, such as use ofinanimate means to actuate the array of plungers. For example, ratherthan employing animate force to work a wing nut, thumb wheel, or drivescrew, the plungers can be actuated by a motor driven plunger. In suchembodiment a motor can be placed in arrangement with the housing 50 to,for example, cause bidirectional rotation of the housing with respect tothe embodiment disclosed in FIGS. 6-8. Likewise a motor can be affixedto gears as in the thumb wheel embodiment to actuate raising andlowering the plungers. Further, as one of ordinary skill in the artwould understand, the device can include indicators to provide theoperator notice when the plungers have reached their respective maximumof travel into or away from the wells 31.

Still further embodiments include use of any variety of energizingparameters including use of monopolar and/or bipolar pulses in any form,including without limitation, trains of pulses, exponential decayingpluses, square pulses, etc. Further still, the invention can use pulseshaving a field strength of between 10 and 1500 V/cm. Moreover, theinvention contemplates the capacity to energize each electrode of thearray individually or together with any combination of the remainingelectrodes. For example, in one embodiment, any 4 adjacent electrodescan be pulsed such that two are positive and an opposing pair ofelectrodes are both negatively charged. Such pulsing provides “opposedpair” pulsing as disclosed in U.S. Pat. Nos. 5,993,434 and 5,702,359. Inyet other pulsing formats, single pairs of electrodes can be energizedto impart an electroporating pulse of energy to the tissue until alladjacent pairs of electrodes are energized at least once. Although notshown in the figures, it is to be understood that the invention deviceas disclosed in its various embodiments includes individually addressedelectrode needles. This is accomplished by incorporating withinsubstrate 22 such as by sandwich technique (i.e., substrate 22 cancomprise a two layer construction with electric leads placed on a gridin between the two layers) electric lead connections running from theelectrodes to the side of the device (as depicted as leads 24)terminating in plug 25 having multiple pin connector capability.

Example IV

In this example, a series of experiments were performed in mice whereintumor recurrence rates were determined after surgery alone or aftersurgery and EPT. Specifically, this experiment comprises an in vivostudy to establish the efficacy of EPT on surgical margin treatment innude mice after subcutaneous introduction of HT-29 human colon carcinomacells and resection of the tumors that arise from such introduction. Theexperiment was conducted under humane conditions with respect totreatment of the test animals.

The experimental design was as follows. Normal Athymic Nude female mice4-6 weeks of age were placed in 8 groups of six mice each, except forGroup 1 which comprised 10 animals and Group 3 comprising 7 animals. TheHT-29 cell line is derived from a human colon carcinoma and is anaggressive fast-growing tumor in mice. In each group the animalsreceived HT-29 human tumor cells in the form of two adjacent inocula of5×10⁶ cells using a 21 gauge needle and syringe. The HT-29 cell line wasobtained from ECACC (#HTB-38), grown in McCoy's 5a medium (modified)with 1.5 mM L-glutamine adjusted to contain 2.2 g/L sodium bicarbonate,90%, and fetal bovine serum, 10%. Cells were divided 1:2 to 1:4 ongrowth cycles until sufficient cells were available to use in thisexperiment.

Following inoculation the tumors became established at the site of theinoculations. The tumor volume on each animal was monitored regularlyand calculated using the formula: Tumor volume=(a²×b/2) where ‘a’ is thesmallest diameter and ‘b’ is the largest diameter perpendicular to ‘a’.Tumors were measured three times per week starting from the day thetumors were palpable by hand. The tumors were allowed to progress untilthe size was between 500 and 1500 cubic millimeters. Surgical tumorremoval, partial removal, or no removal was then performed inassociation with either EPT or no EPT. In these experiments EPT meanstreatment with Bleomycin and electroporation (EP). The EP, whereperformed, was conducted with one pulse cycle (i.e., 6 pulses at 4 Hz)per electrode insertion using a standard MedPulser generator(Genetronics, Inc. San Diego, Calif.) and an applicator with adisposable six needle array of 1 cm diameter, a distance betweenelectrodes of 0.86 cm, and 1 cm needle length. Each square pulse had aduration of 100 usec and an applied voltage of 1500 V. For Groups 4-8and 10 and 11, after intratumoral injection of Bleomycin the needleelectrode array was inserted in such a way as to encompass the tumorwithin the needle array or, in the case of the largest tumors, such thatthe needles penetrated the edge of the tumor. The needle array wasinserted percutaneously and essentially perpendicular to the surface ofthe flank of the mouse carrying the tumor. Alternatively, in those caseswhere the tumor was excised first, followed by i.v. injection ofBleomycin and then by EP, the needle electrode array was insertedpercutaneously encompassing the surgical wound area (Groups 5-8).

Where Bleomycin was used in the experiment, it was injected at 4 U/mlsaline (1 U=1 mg) as uniformly as possible into tumor and margintissues, as indicated, at a dose of 0.25 ml/cm³ volume of tissue to betreated. Alternatively, where indicated, a corresponding amount ofBleomycin was injected into the tail vein.

The experiment used 8 cohorts of at least 6 mice each. Each cohort wassubjected to a treatment regimen of a combination of any of EP,Bleomycin, saline instead of Bleomycin, complete tumor excision, partialtumor excision, and no tumor excision. Tables II and III disclose thetreatment regimens.

TABLE II Excision Bleomycin Remove Partially No Intra- entire removeremoval veneous Group # Mouse # tumor tumor of tumor delivery EP 1 1-1 XX X 1-2 X X X 1-3 X X X 1-4 X X X 1-5 X X X 1-6 X X X 1-7 X X X 1-8 X XX 1-9 X X X  1-10 X X X 2 2-1 X Saline Sham 2-2 X Saline Sham 2-3 XSaline Sham 2-4 X Saline Sham 2-5 X Saline Sham 2-6 X Saline Sham 3 3-1X X X 3-2 X X X 3-3 X X X 3-4 X X X 3-5 X X X 3-6 X X X 3-7 X X X 4 4-1X Saline Sham 4-2 X Saline Sham 4-3 X Saline Sham 4-4 X Saline Sham 4-5X Saline Sham 4-6 X Saline Sham

For experiments on animals listed in Table II, the animals wereanaesthetized, followed by removal or partial removal of the tumor asindicated, followed in turn by intraveneous (i.v.) injection ofBleomycin or saline. The Bleomycin dose was dependent on tumor size asdescribed above. After 3 to 4 minutes, the animals indicated weretreated with EP followed by closure of the wound with clips and surgicaladhesive. In cohort 1, the entire tumor was surgically removed (see, forexample FIGS. 12B and C) and the animals treated with Bleomycin and EP.In cohort 2, the tumors were removed from the animals but only salinewas injected instead of Bleomycin and no EP was performed. Only a “sham”EP procedure was performed in that the electrode array was inserted intothe tissue but the electrodes were not pulsed. In cohort 3, the tumorswere partially (at least 95%) removed followed by Bleomycin injectionand electroporation. In cohort 4, the tumors were again partiallyremoved followed by saline injection and sham EP.

TABLE III Excision Bleomycin Remove Partially No Intra- entire removeremoval tumoral Group # Mouse # tumor tumor of tumor delivery EP 5 5-1 XX X  5-2 X X X  5-3 X X X  5-4 X X X  5-5 X X X  5-6 X X X  6 6-1 X X X 6-2 X X X  6-3 X X X  6-4 X X X  6-5 X X X  6-6 X X X  7 7-1 X X X  7-2X X X  7-3 X X X  7-4 X X X  7-5 X X X  7-6 X X X  8 8-1 X X X* 8-2 X XX* 8-3 X X X* 8-4 X X X* 8-5 X X X* 8-6 X X X*

In Table III the cohort animals were anaesthetized followed byintratumoral administration of Bleomycin solution at a dose and volumeas described above. EP was performed 10 minutes after drugadministration followed by tumor removal or partial tumor removal 15minutes later, followed in turn by wound closure. In cohort 8, EP wasperformed incompletely (denoted by * in Table III) in that the needlearray electrodes were placed such that effective electroporation onlyoccurred in about 75% of the tumor and associated margin tissue. Inother words, the EP treatment was offset with respect to the tumor.

In addition to the above experiments, we also increased the time betweencompletion of EPT and tumor excision to assess both gross consistency oftumor tissue and evaluate it histologically. For this purpose, threeadditional cohort Groups 9, 10, and 11 were used. The experimentalregimen is shown in Table IV.

TABLE IV Excision Complete tumor Group # Mouse # removal Bleomycin EP 9 9-1 X  9-2 X  9-3 X 10 10-1 X (at 2 hr) X X 10-2 X (at 2 hr) X X 10-3 X(at 2 hr) X X 11 11-1 X (at 24 hr) X X 11-2 X (at 24 hr) X X 11-3 X (at24 hr) X XThe animals of Group 9 were anaesthetized followed by complete excisionof the tumors. The tumors were examined for consistency and preserved informalin for later histological evaluation, as were the tumor tissues ofcohorts 10 and 11. Anaesthetized animals of cohort 10 were subjected tointratumoral administration of Bleomycin solution at a dose and volumeas described above. EP was performed 10 minutes after drugadministration. Tumors were excised 2 hrs after EP. Animals of cohort 11were treated as those in cohort 10 except that their tumors werecompletely removed at 24 hrs after EP. All wounds were closed asdescribed above.

Results

With respect to the above cohorts 1 to 8 we examined the rate ofrecurrence of the tumors in the test animals three weeks aftertreatment. As disclosed in Table V and FIG. 15, the majority of animalsreceiving Bleomycin and EP were protected from tumor recurrence. (seedescription of FIG. 15 for definitions of abbreviations therein).

TABLE V Route of administration Group Tumor excision of Bleomycin % #complete partial none EP i.v. intratumoral efficacy* 1 + + + 90 5 + + +100 7 + + + 100 6 + + + 80 3 + + + 67 8 + +/−** + 67 2 + − 0 4 + − 0 *%efficacy is based on the number of test animals in each Group that didNOT experience tumor growth post treatment **+/− represents a partialEPT procedure wherein the treatment was off-centered from thetumor/tumor bed.

Specifically, Groups 1 to 8 provide results indicating that treatment oftumor bed tissue provides an unexpected and surprising benefit inprotecting the animal from recurrent tumors at the site of thetreatment. As shown by the data, excision alone is not effective,whether the tumors were completely or partially removed. This is likelydue to the aggressive nature of the tumor type used which left invasivetumor segments and/or micrometastases at or close to the tumor sitedespite careful surgical removal of the tumor correlating to similaraggressive tumors in man. This mimics situations encountered in humansurgical therapy although recurrence rates in humans are generally inthe 10 to 40% range and not as high as observed in this mouseexperiment. Importantly, as exhibited by the results of the partialtumor removal, use of EPT with Bleomycin is associated with substantialreduction in tumor recurrence even if the surgical procedure failed (inthis case on purpose) to excise all of the tumor mass. Further, theroute of administration does not appear to play a significant role inefficacy in that administration by i.v. and i.t., respectively, producedsimilar results. Further still, the efficacy effect was surprisinglyprovided even after a 15 minute interval between completion ofBleomycin-EP treatment and tumor excision. The data are furthersupportive of the treatment methods in effectively treating microtumorseeding in the tissue in that the experiment wherein EP was onlypartially performed without tumor removal, i.e. the effective electricalfield was off-centered from the tumor. Here, there was still 67%efficacy indicating there was at least a microregional effect caused bythe EPT. In this example study cohorts for testing Bleomycinadministration alone without EP or vice versa (i.e. EP withoutBleomycin) were not included as Bleomycin administration without EP isknown to have no significant antitumoral activity.

With respect to the tumor cells introduced into the test animals, weallowed the tumor masses to grow to relatively large size. See FIGS.11A, 12A, 13A and 14A which represent tumors in test animals prior totreatment. FIGS. 11B and 12B represent examples of surgical tumorremoval at the stage of having the tumor exposed before the finalexcision. FIGS. 11C, 12C, 13B, and 14B represent examples of the woundafter complete tumor removal prior to Bleomycin-EPT (FIG. 12C) orsaline-sham EP treatment (FIG. 11C), or after partial tumor removalprior to saline-sham treatment (FIG. 14B), or 15 minutes after i.t.Bleomycin-EPT (FIG. 13B).

FIG. 11D shows recurring tumor after complete tumor excision andsaline-sham EP treatment and FIG. 14C shows recurring tumor afterpartial tumor excision and saline-sham EP treatment. FIGS. 12D and 13Cshow tumor sites free of tumor after tumor excision and treatment withi.v. Bleomycin and EP, or treatment with i.t. Bleomycin and EP withsubsequent partial tumor excision, respectively. These procedures haveadvantages over present standard procedures that treat cancers withanticancer drug i.t. or i.v. Excision of the tumor, either prior to orafter drug-EPT treatment prevents formation of a large necrotic mass atthe tumor site which the body has to resorb or otherwise eliminate,which empirically takes at least several weeks and enhances theprobability of complications. Removal of the tumor prior to or afterdrug-EPT is likely to expedite wound healing and reduce potentialcomplications. Further with this invention methodology, bothintratumoral and intravenous injection of drug can be used.

With respect to cohorts 9-11, we observed that the consistency of thetumor changed progressively with time after treatment. When the tumorwas excised two hours after treatment (cohort 10) its consistency wassofter, less well defined and it leaked edemic fluid but relativelylittle blood. Tumors excised 24 hrs after treatment (cohort 11) hadcompletely softened to a consistency sometimes referred to asliquefactive necrosis. At that time point surgical excision was somewhatdifficult because the boundaries of the tumor were ill defined ascompared to the solid tumor tissue mass of cohort 9 animals.Interestingly, we also noticed a softening of the tumor even 15 minutesafter treatment when we removed tumors from cohorts 5 and 7, which madeit slightly more difficult to perform surgical removal with well definedboundaries than in the case of untreated tumors. However, this did notaffect the success of the treatment since both cohorts 5 and 7 showed notumor recurrence. These observations provide proof of the opportunity ofusing drug, such as Bleomycin or other anticancer agent, in combinationwith EPT as adjuvant or tumor debulking therapy that can additionally beused alone or in conjunction with other cancer therapies. For example, atumor could be treated with Bleomycin-EPT, the disintegrated tumormaterial be removed by a simple minimally invasive procedure, and ifdesired, followed by conventional treatments such as radiation orchemotherapy, or other tumor therapy modalities. Easy removal of theliquefied tumor material can also allow the wound to heal faster andwith fewer complications, especially in the case of large tumors.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the spirit and scopeof the invention. More specifically, the described embodiments are to beconsidered in all respects only as illustrative and not restrictive. Allsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit and scope of the invention asdefined by the appended claims.

All patents, patent applications, and publications mentioned in thespecification are indicative of the levels of those of ordinary skill inthe art to which the invention pertains. All patents, patentapplications, and publications, including those to which priority oranother benefit is claimed, are herein incorporated by reference to thesame extent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising”, “consisting essentially of”, and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that use of such terms andexpressions imply excluding any equivalents of the features shown anddescribed in whole or in part thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

What is claimed is:
 1. A method of reducing recurrence of tumor cellgrowth in a mammalian tissue, the method comprising: (a) providing asource for applying an electroporative electric pulse to an unexcisedtumor mass and a margin tissue around the unexcised tumor mass; (b)providing an agent capable of reducing tumor cell growth; (c) injectingthe agent into the unexcised tumor mass and the margin tissue around theunexcised tumor mass; and (d) applying the electroporative electricpulse by the source to the unexcised tumor mass and the margin tissuearound the unexcised tumor mass, thereby delivering the agent into cellsof the unexcised tumor mass and the margin tissue around the unexcisedtumor mass; wherein recurrence of tumor cell growth in the mammaliantissue is reduced.
 2. The method of claim 1, wherein the agent isselected from the group consisting of Bleomycin, cisplatin, apolypeptide, an antibody, an RNAi, an antisense nucleic acid, anexpressible gene encoding a therapeutically active polypeptide, achemokine, and a cytokine.
 3. The method of claim 2, wherein the RNAi,the antisense nucleic acid, or the expressible gene is formulated inpoly-L-glutamate solution.
 4. The method of claim 1, wherein the tumorcell is selected from the group consisting of: a cancer cell incutaneous tissue, a cancer cell located on the head or neck of a mammal,a squamous cell carcinoma, a colon carcinoma, and a melanoma cell. 5.The method of claim 1, wherein the electroporative electric pulsecomprises a pulse having a nominal field strength selected from thegroup consisting of: between 800 and 1500 V/cm, between 600 and 1500V/cm, between 600 and 1400 V/cm, between 200 and 800 V/cm, between 1 and600 V/cm, between 200 to 600 V/cm, between 400 and 600 V/cm, 1200 V/cm,and 1500 V/cm.
 6. The method of claim 1, wherein the source comprises:an array of a multiplicity of electrodes capable of channeling a fluidvolume from at least one variable volume reservoir to the unexcisedtumor mass and the margin tissue around the unexcised tumor mass whenthe multiplicity of electrodes are positioned in the unexcised tumormass and the margin tissue around the unexcised tumor mass, wherein saidmultiplicity of electrodes can be further energized in a predeterminedformat selected from the group consisting of energizing single pairs ofelectrodes, energizing opposed pairs of electrodes, energizing aselected portion of said electrodes sequentially, and energizing aselected portion of said electrodes simultaneously.
 7. The method ofclaim 1, wherein the source comprises: (a) an array of a multiplicity ofelectrodes comprising lumens running therethrough for transporting afluid medium; (b) an array of compartments capable of containing a fluidmedium and in fluid communication with said lumens; (c) an array ofplungers corresponding to said array of compartments wherein a plungeris fit into each compartment of said array of compartments and whereinsaid array of plungers are capable of slidably adjustable positioning intheir respective compartments; (d) an actuator for slidably adjustingpositioning of said plungers; and (e) a source of electrical energyconnectable to said multiplicity of electrodes for imparting to saidmultiplicity of electrodes the electroporative electric pulse.
 8. Themethod of claim 7, wherein the actuator is a thumb wheel.
 9. The methodof claim 7, wherein the actuator is a wing nut.
 10. The method of claim7, wherein the actuator is an electric motor.
 11. The method of claim 7,wherein the actuator is screw driven.
 12. The method of claim 1, whereinthe unexcised tumor mass has been liquified after step (d).
 13. Themethod of claim 12, wherein the unexcised tumor mass has undergoneliquefactive necrosis after step (d).
 14. The method of claim 13,wherein the unexcised tumor mass has been debulked after step (d). 15.The method of claim 1, wherein the unexcised tumor mass has beensoftened after step (d).
 16. The method of claim 15, wherein theunexcised tumor mass is softened within about 24 hours.
 17. The methodof claim 16, wherein the unexcised tumor mass is softened within about15 minutes.
 18. The method of claim 1, further comprising administeringone or more additional cancer therapies.
 19. The method of claim 18,wherein the one or more additional cancer therapies is selected from thegroup consisting of: radiation, chemotherapy, and antibody therapy. 20.The method of claim 1, wherein (d) further comprises treating microtumorseeding in the margin tissue.